Conventional reversed phase silica columns (e.g., ODS) are widely used as general-purpose stationary phases for chromatographic separations (Neue, “HPLC Columns—Theory, Technology, and Practice,” WILEY-VCH, New York, 1997, 183-203). However, some drawbacks, including, for example, “phase collapse”,(i.e., dewetting) in highly aqueous environments, weak retention of ionic compounds and residual silanol activity which leads to peak tailing of basic analytes prevent employment of convention reverse phase silica columns in certain applications.
Polar-embedded phases improve the peak shape of basic analytes and enable operation of reverse phase HPLC columns in highly aqueous environments (O'Gara et al., LC-GC 2001, 19 (6):632-641). Polar embedded phases are primarily hydrophobic but have hydrophilic groups near the substrate surface. Commonly used polar groups include, for example, amides, ureas, ethers and carbamates. In general, polar-embedded phases provide superior peak shapes of basic analytes and are more compatible with highly aqueous environments when compared to general purpose reverse phases. Further, polar embedded phases often have selectivities which are substantially different from those exhibited by conventional C-18 packings. However, ionic compounds such as small hydrophilic organic acids or inorganic ions are poorly separated, if at all, by chromatography on polar embedded phases.
Typically, ion-exchange chromatography is used to separate ionic or ionizable compounds such as proteins, nucleic acids, inorganic ions, small organic acids, etc. (Neue, supra, 224-249). However, since hydrophobic molecules are poorly retained on most ion exchange resins, ion-exchange chromatography is rarely used in conventional HPLC for the separation of organic molecules.
Ion-pairing chromatography is another method for separating ionic or ionizable compounds (Neue, supra, 209-211). Here, hydrophobic ionic compounds, typically comprised of an alkyl chain with an ionizable terminus, are added to the mobile phase while the stationary phase is conventional reversed-phase medium. Generally, retention of neutral analytes is nearly unaffected, while analytes with charges complementary to the ion-pairing reagent are retained for a longer period of time and analytes with the same charge as the ion-pairing reagent are retained for a shorter period of time. As is known to the skilled artisan, retention of charged analytes may be affected by a variety of factors including, for example, the type and concentration of the ion-pairing reagent, ionic strength and the pH of the mobile phase. Limitations of ion-pairing chromatography include long column equilibration times and the quantity of solvent and time needed to elute the ion-pairing reagent from the column. Further, the presence of ion-pairing reagent complicates the composition of the mobile phase and can interfere with many detection methods, such as, for example, electrochemical detection modes.
Yet another method for separating ionic or ionizable compounds is mixed-mode chromatography, which combines aspects of ion-exchange chromatography and conventional reverse phase chromatography. For example, commercially available aminopropylsilyl bonded phase, modified with different hydrophobic organic acids to provide weakly hydrophobic anion-exchange supports, has been used to separate oligonucleotides (Bischoff et al., Journal of Chromatography 1983, 270:117-126). Here, ion-exchange is the primary mode of separation because the hydrophobicity of this resin is due to a three-carbon linker.
Other mixed-mode supports for liquid chromatography which were used to separate nucleic acids have been made by functionalizing anion-exchange surfaces with hydrophobic groups (Bischoff et al., Journal of Chromatography 1984, 296:329-337). Although this method can be used to modulate the hydrophobicity of the stationary phase, the slightly different amounts of ion-exchange and hydrophobic sites introduced onto the surface during each functionalization negatively affect support reproducibility. Mixed-mode polymeric resins useful for separating proteins and peptides have been prepared by activation of hydroxylated polymer surfaces with carbonyldiimidazole or epichlorohydrin, followed by reaction with primary amines containing ion-exchange functionality (Burton et al., U.S. Pat. No. 5,945,520). Ion-exchange matrices based on porous magnetic silica particles have used to separate nucleic acids, such as plasmid DNA, chromosomal DNA or RNA from contaminants including proteins, lipids, cellular debris, etc. (Smith et al., U.S. Pat. No. 6,310,199). Finally, a family of mixed-mode HPLC columns, which have ion-exchange functionality embedded between the silica surface and an alkyl chain and thus provide both ion-exchange and hydrophobic retaining sites have become commercially available (SIELC Technologies, Prospect Heights, ILL).
Despite the advances in mixed-mode chromatography, supra, novel silane compounds which have both hydrophobic and ionic functionality, substrates functionalized with these new silane compounds and the use of these novel functionalized substrates in mixed mode chromatography are needed to provide for controlled retention of both ionizable and neutral compounds. Ideally, the novel functionalized substrates will retain ionic or ionizable compounds in the absence of ion-pairing reagents, allow for simultaneous analysis and separation of inorganic ions and organic compounds, mask the effect of ion-exchange groups on the residual interaction of basic analytes with surface silanols and increase resistance of developed stationary phases to dewetting in 100% aqueous media.